Saxatilin-fc fusion protein and use thereof

ABSTRACT

The present invention relates to a saxatilin derivative having an increased half life and a use thereof. The saxatilin derivative of the present invention has thrombolytic ability similar to that of saxatilin, which is the mother protein, a remarkably increased protein half life, and efficiently dissolves, for long period of time, blood clots already formed in blood vessels of an animal model with a FeCl 3 -induced carotid by using the same. Therefore, a composition containing, as an active ingredient, the saxatilin derivative of the present invention does not cause reocclusion after penetration and effectively opens to microvessels, and is thus very useful for treating angiostenosis or occlusive diseases (for example, cerebrovascular diseases, cardiovascular diseases, arteriosclerotic vascular diseases, coronary artery diseases, and peripheral vascular diseases).

FIELD

The present invention was made with the support of the Ministry ofHealth and Welfare, Republic of Korea, under Project No. A085136, whichwas conducted in the program titled “Leading Characterization Researchand Development Business” in the project named “LeadingCerebro-cardiovascular Diseases Fusion Research Business Consortium”, bythe Industry-Academic Cooperation Foundation, YONSEI University, undermanagement of the Korea Health Industry Development Institute, from 1Dec. 2012 to 30 Nov. 2013.

This application claims priority to and the benefit of Korean PatentApplication Nos. 10-2013-0090468 and 10-2014-0097461, filed in theKorean Intellectual Property Office on 30 Jul. 2013 and 30 Jul. 2014,the entire contents of which are incorporated herein by reference.

The present invention relates to a saxatilin derivative having anincreased half-life and a use thereof.

BACKGROUND

Most strokes are caused by thromboembolic occlusion in major or smallerintracerebral arteries (Wardlaw, Murray et al. 2009). In ischemicstroke, rapid thrombolysis is the only established therapeutic option tosuppress the onset of unavoidable complete infarction (1995; Choi andBateman et al. 2006). Treatment through the intravenous administrationof recombinant tissue plasminogen activator (r-tPA) is currently theonly approved therapy for ischemic stroke, which can be made within 4.5hours after the onset of ischemic stroke (Caplan, Mohr et al. 1997; andLopez-Yunez, Bruno et al. 2001). However, more than half the patientsfailed to achieve successful recanalization after thrombolytic treatment(Rha and Saver 2007; and Lee et al. Stroke 2007; 38:192-193]. Even ifoccluded arteries are successfully recanalized by the thrombolytictreatment, these benefits are again degraded due to the risks ofreperfusion injury (Hallenbeck and Dutka 1990), intracerebral hemorrhage(Adams, Adams et al. 2005), and reocclusion (Heo, Lee et al 2003). Inaddition, rt-PA was reported to have neurotoxicity (Chen and Strickland1997; Wang, Tsirka et al. 1998; Nicole, Docagne et al. 2001; Yepes,Sandkvist et al. 2002; and Matys and Strickland 2003).

Although the recanalization strategy has proven its efficacy, it causedlimited applicability and potential adverse effects, and thus there havebeen efforts to develop new thrombolytic agents having better effectsthan rt-PA. These efforts include variants of t-PA, plasminogenactivators derived from animal sources, and microplasmin. Theabove-cited drugs have the following purposes: (a) enhancing fibrinspecificity; (2) extending plasma half-life; (3) reducing inhibitoryability by plasminogen activator inhibitor-1; and (d) avoidingneurotoxicity. Several drugs have completed clinical trials, and somedrugs are being studied for their efficacy. These drugs target fibrin inthe thrombi, and thrombolytic agents, such as rt-PT and urokinase,correspond to these drugs. However, the thrombi are formed by aplatelet-fibrinogen interaction. Thrombin, leukocytes, and erythrocytesare also components of the thrombus. The resistance of the thrombi tothe thrombolytic agents targeting fibrin is one of the main causes oflow recanalization rates in stroke patients, which may occur morecommonly in occlusion by platelet-rich thrombi. In this regard, thetreatment targeting platelets may be an option or additive to thetreatment targeting fibrin for improved thrombolytic efficacy.

Platelet glycoprotein (GP) IIb/IIIa, a member of the integrin family,exists on the surface of the platelet membrane at high density (Shattiland Ginsberg 1997). The GPIIb/IIIa receptor mediates the final stage ofthe platelet aggregation pathway by specifically binding to fibrinogen(Phillips, Charo et al. 1988). Therefore, targeting the plateletGPIIb/IIIa receptor has been the mainstay for the development of drugsacting on the platelets. Many platelet GPIIb/IIIa antagonists have beendeveloped, which include the Fab fragment of a human-mouse chimericantibody against GP IIb/IIIa (abciximab), nonpeptide analogues of RGDpeptides (tirofiban and lamifiban), and cyclic heptapeptide disintegrincontaining KGD motif (eptifibatide) (Seitz, Meisel et al. 2004;Abou-Chebl, Bajzer et al. 2005; and Eckert, Koch et al. 2005). TheseGPIIb/IIIa antagonists have been effective for patients with unstableangina, acute myocardial infarction, and percutaneous treanfemoralcoronary angioplasty (PTCA) and patients receiving stent. In stroke,abciximab failed to show efficacy in patients who were treated 5 to 6hours after the symptom onset (Adams, Effron et al. 2008). However,GPIIb/IIIa antagonists lyse the thrombi in stroke patients withreocclusion, and are effective for selected patients (Heo, Lee et al.2003; Seitz, Hamzavi et al. 2003; Seitz, Meisel et al. 2004; Eckert,Koch et al. 2005; and Chen, Mo et al. 2007).

Saxatilin, which is a novel disintegrin that is purified and cloned fromKorean snake venom, has the tripeptide sequence Arg-Gly-Asp (RGD), whichis a recognition site of disintegrin to the platelet GPIIb/IIIa receptor(Hong, Koh et al. 2002; and Hong, Sohn et al. 2002). It has been knownthat saxatilin has strong inhibitory effects on platelet aggregation(Hong, Koh et al. 2002) and platelet activation (Jang, Jeon et al.2007), and thus interrupts the generation of thrombus.

Throughout the entire specification, many papers and patent documentsare referenced and their citations are represented. The disclosure ofthe cited papers and patent documents are entirely incorporated byreference into the present specification and the level of the technicalfield within which the present invention falls, and the details of thepresent invention are explained more clearly.

DETAILED DESCRIPTION Technical Problem

The present inventors have endeavored to develop a thrombolytic agent,which can recanalize a partially or completely occluded blood vessel byeffectively suppressing the generation of thrombi or lysing the alreadygenerated thrombi. As a result, the present inventors validated that arecombinant protein (or a saxatilin derivative), which is composed ofdisintegrin, specifically, saxatilin, derived from Korean snake venom,and an immunoglobulin Fc fragment, has similar thrombolytic activity tothe saxatilin protein as well as a significantly increased proteinhalf-life, enables very efficient lysis of the thrombi, which werealready generated in the blood vessel in FeCl₃-induced carotid arteryanimal models, and can maintain its activity for a longer time than theparent protein, saxatilin, and thus the present inventors completed thepresent invention.

Accordingly, an aspect of the present invention is to provide asaxatilin derivative.

Another aspect of the present invention is to provide a nucleotidesequence encoding the saxatilin derivative.

Still another aspect of the present invention is to provide arecombinant vector including the nucleotide sequence.

Still another aspect of the present invention is to provide cellstransfected with the recombinant vector.

Still another aspect of the present invention is to provide acomposition for thrombolysis.

Still another aspect of the present invention is to provide apharmaceutical composition for preventing or treating stenosis of bloodvessel or occlusive disease.

Still another aspect of the present invention is to provide a method forpreventing or treating stenosis of blood vessel or occlusive disease.

Still another aspect of the present invention is to provide a use of asaxatilin derivative for preventing or treating stenosis of blood vesselor occlusive disease.

Other purposes and advantages of the present disclosure will become moreobvious with the following detailed description of the invention,claims, and drawings.

Technical Solution

In accordance with an aspect of the present invention, there is provideda saxatilin derivative including saxatilin, which is composed of theamino acid sequence of SEQ ID NO: 2, conjugated to an immunoglobulin Fcregion.

In accordance with another aspect of the present invention, there isprovided a nucleotide sequence encoding the foregoing saxatilinderivative.

In accordance with another aspect of the present invention, there isprovided a recombinant vector, including: (a) the foregoing nucleotidesequence; and (b) a promoter operatively linked to the nucleotidesequence.

In accordance with still another aspect of the present invention, thereis provided a cell or transformant transinfected with the foregoingrecombinant vector.

The present inventors have endeavored to develop a thrombolytic agent,which can recanalize a partially or completely occluded blood vessel byeffectively suppressing the generation of thrombi or lysing the alreadygenerated thrombi. As a result, the present inventors validated that arecombinant protein (or a saxatilin derivative), which is composed ofdisintegrin, specifically, saxatilin, derived from Korean snake venom,and an immunoglobulin Fc fragment, has similar thrombolytic activity tothat of the saxatilin protein as well as a significantly increasedprotein half-life, enables very efficient lysis of the thrombi, whichwere already generated in the blood vessel in FeCl₃-induced carotidartery animal models, and can maintain its activity for a longer timethan the parent protein, saxatilin.

The saxatilin derivative of the present invention, which is a fusionprotein of saxatilin (SEQ ID NO: 1 and SEQ ID NO: 2) and Fc, is composedof saxatilin, including an amino acid sequence of SEQ ID NO: 2, and animmunoglobulin Fc region. The saxatilin derivative can efficiently breakup the thrombus through a principle in which the saxatilin derivativecompetitively binds to an integrin existing in the thrombus(specifically, glycoprotein (GP) Ilb-Ilia on a surface of plateletsconstituting the thrombus), thereby separating the platelets and thelike from constituents of the thrombus, such as fibrinogen.

According to a certain embodiment of the present invention, thesaxatilin derivative of the present invention has similar thrombolyticactivity to saxatilin, which is the parent protein thereof.

In an embodiment of the present invention, the saxatilin derivative ofthe present invention has an IC₅₀ value of 100-500 nM, and preferably100-250 nM, with respect to platelet aggregation.

Specifically, the IC₅₀ values of saxatilin as the parent protein,Fc-saxatilin, and saxatilin-Fc, were 150 nM, 196 nM, and 362 nM,respectively (see: FIG. 4). As used herein, the term “IC₅₀ value (halfmaximal inhibitory concentration)” refers to an indicator that evaluatesthe efficacy of a compound inhibiting biological or biochemicalactivity, and offers a quantitative concentration measurement value atwhich a particular drug or other substances (e.g., a saxatilinderivative of the present invention) inhibit a given biological process(e.g., thrombolysis) or components of the process (e.g., an enzyme orcell receptor) by half. It is commonly used as a measure of antagonistdrug potency in pharmacological research. Meanwhile, thrombolyticactivity may be measured by using various methods known in the art: forexample, fibrin plate assay (Astrup A and Mullertz S., The fibrin platemethod for estimating fibrinolytic activity. Arch Biochem Biophys 40:346-351(1952)); the detection of D-dimer showing the breakage ofplatelets (U.S. Patent Publication No. US 2009/0305301 A1); the use ofradioisotope (PCT Patent Publication No. WO 1994/022494); themeasurement of thrombus weight in the blood vessel; and the measurementof turbidity of powder released after thrombolysis, but are not limitedthereto.

In an embodiment of the present invention, the saxatilin derivative ofthe present invention has a binding affinity (dissociation constant(Kd)) of 1×10⁻⁸ to 1×10⁻¹° M to integrin α_(M)β₂ existing inneutrophils.

In an embodiment of the present invention, the saxatilin derivative ofthe present invention has a binding affinity (dissociation constant(Kd)) of 1×10⁻⁸ to 1×10⁻¹° M to integrin α_(L)β₂ existing inneutrophils.

According to a certain embodiment of the present invention, thethroughput of the saxatilin derivative of the present invention is 1-20mg/kg, more specifically, 3-15 mg/kg, and most specifically, 5-10 mg/kg.According to the present invention, the saxatilin derivative of thepresent invention showed an effect on the recanalization of thecompletely occluded blood vessel at a small dose (see FIGS. 6A to 6B).As can be confirmed in FIG. 6, the treatment with 130 nmol/kg saxatilin,Fc-saxatilin, or saxatilin-Fc showed a blood flow recovery effect ofabout 30% in the recanalization of the completely occluded blood vessel.Further, the saxatilin treatment group showed reocclusion atapproximately 90 min, but the Fc-saxatilin or saxatilin-Fc treatmentgroup showed a continuous recanalization effect.

According to a certain embodiment of the present invention, thesaxatilin derivative of the present invention may be administered orallyor parentally, specifically parentally, and examples of the parentaladministration may include bolus injection, intravenous injection,intra-arterial injection, intramuscular injection, intraperitonealinjection, topical administration, dermal administration, etc., and mostpreferably, the saxatilin derivative of the present invention may beadministered by bolus injection.

In addition, the drug having a vascular recanalization effect (e.g., thesaxatilin derivative of the present invention), preferably, has asuitable retention time, and specifically, a retention time ofapproximately 10 min is clinically most suitable and effective.

According to a certain embodiment of the present invention, theimmunoglobulin Fc region contained in the saxatilin derivative of thepresent invention is conjugated to the N-terminal or C-terminal ofsaxatilin, and more specifically, conjugated to the N-terminal ofsaxatilin.

According to a certain embodiment of the present invention, thesaxatilin derivative of the present invention further includes a leadersequence at the N-terminal thereof.

Conventionally, the fusion protein having the immunoglobulin Fc regionbinding to a target protein exhibits lower activity or a smallerhalf-life increase effect when compared with the parent protein.Whereas, the saxatilin derivative of the present invention not only hadsimilar thrombolytic activity to the parent protein, but also showed asignificantly increased half-life compared with the parent protein (seeFIGS. 5A to 5C). Specifically, the half-lives of Fc-saxatilin andsaxatilin-Fc of the present invention were 8.6 min and 12.5 min,respectively, which were much longer than the half-life (2.0 min) of theparent protein saxatilin.

According to a certain embodiment of the present invention, thesaxatilin derivative of the present invention has a half-life, which isincreased by at least 4-fold, more specifically, about 4- to 6.5-fold,when compared with the parent protein saxatilin.

As used herein, the term “Fc region” refers to the carboxyl terminalportion of an immunoglobulin chain constant region, specifically, animmunoglobulin heavy chain constant region or a part thereof. Forexample, the immunoglobulin Fc region that may be used in thepreparation of the saxatilin derivative of the present invention, mayinclude: (a) CH1 domain, CH2 domain, and CH3 domain; (b) CH1 domain andCH2 domain; (c) CH1 domain and CH3 domain; (d) CH2 domain and CH3domain; or (e) a combination of two or more domains and a immunoglobulinhinge region.

According to a certain embodiment of the present invention, theimmunoglobulin Fc region that may be used in the preparation of thesaxatilin derivative of the present invention includes a humanimmunoglobulin Fc region (GenBank Accession No., CAA49866.1) andfragments thereof (e.g., SEQ ID NO: 7).

Besides the human immunoglobulin Fc region, amino acid sequences, whichare encoded by the nucleotide sequences disclosed in GenBank and/or EMBLdatabase, for example, other immunoglobulin Fc regions includingAF045536.1 (Macaca fuscicularis), AF045537.1 (Macaca mulatta), AB016710(Felix catus), K00752 (Oryctolagus cuniculus), UO3780 (Sus scrofa),Z48947 (Camelus dromedarius), X62916 (Bos taurus), L07789 (Mustelavision), X69797 (Ovis aries), U17166 (Cricetulus migratorius), X07189(Rattus rattus), AF57619.1 (Trichosurus vulpecula) orAF035195(Monodelphis domestica), or fragments thereof may also be used.

Further, the substitution or deletion of the amino acid sequence in theimmunoglobulin heavy chain constant region may be used in thepreparation of the saxatilin derivative of the present invention. Fcmutants, which are prepared through the substitution or deletion,improve safety, dissolubility, and structural integrity of fusionproteins, and thus are useful in the preparation of optimized fusionproteins.

The present invention provides a nucleotide sequence encoding a fusionprotein (SEQ ID NO: 4 or SEQ ID NO: 6) composed of saxatilin of SEQ IDNO: 2 and the immunoglobulin Fc region of SEQ ID NO: 7, which arespecifically exemplified as SEQ ID NO: 3 and SEQ ID NO: 5, but are notlimited thereto. It would be obvious to a person skilled in the art thatany nucleotide sequence that encodes the fusion protein may be used.

In addition, the fusion protein of the present invention may be taggedwith various detectable indication tags, and the examples of the tag mayinclude a flag, c-Myc, HA, V5, VSV-G, and HSV, but are not limitedthereto. As used herein, the term “tag” refers to a polynucleotidesequence having 3 to 40 amino acid sequences, and gives specific bindingaffinity to the fusion protein of the present invention, a peptide, aprotein ligand (e.g., the fusion protein of the present invention), or anon-peptide ligand. In addition, the tag that may be used in the presentinvention may include a fluorescent tag, a luminescent tag, and achromogenic tag.

The saxatilin derivative of the present invention may be mass-producedat low costs by using a recombinant vector including: (a) a nucleotidesequence encoding the saxatilin derivative; and (b) a promoteroperatively linked to the nucleotide sequence.

As used herein, the term “promoter” refers to a DNA sequence thatregulates the expression of a coding sequence or functional RNA. In therecombinant expression vector of the present invention, a material to beexpressed (i.e., saxatilin-Fc-fused protein)-encoding nucleotidesequence is operatively linked to the promoter. As used herein, the term“operatively linked” refers to a functional linkage between a nucleicacid expression regulating sequence (e.g., a promoter sequence, a signalsequence, or an array at the binding site of a transcription controlfactor) and another nucleic acid sequence, and the regulating sequenceregulates the transcription and/or translation of the another nucleicacid sequence.

The vector system of the present invention can be constructed by variousmethods known in the art, and a specific method thereof is disclosed inSambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press (2001), which is incorporated herein byreference.

In cases where the vector of the present invention uses prokaryoticcells as a host, it generally carries a strong promoter to initiatetranscription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpppromoter, p_(L) promoter, p_(R) promoter, rac5 promoter, amp promoter,recA promoter, SP6 promoter, trp promoter, or T7 promoter), a ribosomebinding site for translation initiation, and a transcription/translationtermination sequence. Here, the bacterial replication origin may beselected from replication origins that are well known in the art to beuseful in the stable bacterial replication of long DNA inserts, andexamples thereof may include ColE1, F-factor, and P1 replicon, but arenot limited thereto. As the bacterial selection marker of the presentinvention, bacterial selection marker genes that are known in the artmay be used. For example, examples of the bacterial selection markergene may include genes that confer resistance to antibiotics, such asampicillin, kanamycin, tetracycline, Zeocin, neomycin, hygromycin, andchloramphenicol, but are not limited thereto. In cases where E. coli isused as a host cell, the promoter and operator region for the tryptophanbiosynthesis pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024(1984))and the leftward promoter from phage λ (p_(L)λ promoter, Herskowitz, I.and Hagen, D., Ann. Rev. Genet., 14:399-445(1980)) may be used asregulating sequences.

In addition, in cases where the recombinant vector of the presentinvention is applied to eukaryotic cells, the promoter can regulate thetranscription of the material to be expressed, of the present invention,and includes promoters derived from mammalian viruses and promotersderived from mammalian cell genomes. Examples thereof may includecytomegalovirus (CMV) promoter, adenovirus late promoter, vaccinia virus7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, EF1alpha promoter, metallothionein promoter, beta-actin promoter, apromoter of human IL-2 gene, a promoter of human IFN gene, a promoter ofhuman IL-4 gene, a promoter of human lymphotoxin gene, and a promoter ofhuman GM-CSF gene, but are not limited thereto.

Preferably, the recombinant vector used in the present inventionincludes a polyadenylation sequence (e.g., bovine growth hormoneterminator or SV40-derived polyadenylation sequence).

The delivery of the vector of the present invention into the host cellmay be conducted by various methods known in the art. For example, incases where the host cells are eukaryotic cells, the delivery may beconducted by the CaCl₂ method (Cohen, S. N. et al., Proc. Natl. Acac.Sci. USA, 9:2110-2114(1973)), the Hannahan's method (Cohen, S. N. etal., Proc. Natl. Acac. Sci. USA, 9:2110-2114(1973); and Hanahan, D., J.Mol. Biol., 166:557-580(1983)), and an electroporation method (Dower, W.J. et al., Nucleic. Acids Res., 16:6127-6145(1988)). In cases where thehost cells are prokaryotic cells, the delivery may be conducted by usingtransduction, electroporation, lipofection, microinjection, particlebombardment, yeast spheroplast/cell fusion used in YAC,Agrobacterium-mediated transformation used in plant cells, or the like.

In addition, the production of animal cells using the recombinant vectorof the present invention may be achieved by a gene transfer method thatis commonly known in the art. For example, the method includeselectroporation, liposome-mediated transfer (Wong, et al., 1980), andretrovirus-mediated transfer (Chen, H. Y., et al., (1990), J. Reprod.Fert. 41:173-182; Kopchick, J. J. et al., (1991) Methods for theintroduction of recombinant DNA into chicken embryos. In TransgenicAnimals, ed. N. L. First & F. P. Haseltine, pp. 275-293, Boston;Butterworth-Heinemann; Lee, M.-R. and Shuman, R. (1990) Proc. 4th WorldCongr. Genet. Appl. Livestock Prod. 16, 107-110), but is not limitedthereto.

According to a certain embodiment of the present invention, theforegoing transgenic animal cells are mammal-derived cells.

According to a certain embodiment of the present invention, when animalcells with a foreign gene are selected, the foreign gene is a selectionmarker, and preferably includes an antibiotic-resistant gene. Theselection marker that is usable in the present invention may be any genethat confers antibiotic activity to eukaryotic cells, and examplesthereof may include neomycin- and kanamycin-resistant genes, but are notlimited thereto.

In accordance with another aspect of the present invention, there isprovided a composition for thrombolysis, the composition containing: (a)a pharmaceutically effective amount of the foregoing saxatilinderivative; and (b) a pharmaceutically acceptable carrier.

In accordance with still another aspect of the present invention, thereis provided a pharmaceutical composition for preventing or treatingvascular stenosis or occlusive disease, the pharmaceutical compositioncontaining the foregoing composition for thrombolysis.

Since the composition of the present invention contains the foregoingsaxatilin derivative of the present invention as an active ingredient,descriptions of overlapping contents between the two are omitted toavoid excessive complication of the specification due to repetitivedescriptions thereof.

The composition of the present invention can be conveniently andefficiently applied to the treatment of a variety of vascular stenosisor occlusive disease by effectively lysing the already generated andagglomerated thrombus. That is, the composition of the present inventioncan be applied to the treatment of vascular stenosis or occlusivedisease through a principle in which the already generated thrombi arelysed to penetrate blood vessels effectively.

As used herein, the term “occlusion” refers to covering the narrowing ofblood vessels due to the complete or partial blocking of the bloodvessel. The degree of occlusion, recited in the present invention, maybe determined based on the measured blood flow. That is, the degree ofocclusion is categorized as a partial occlusion or a complete occlusion.The partial occlusion means the reduction of the baseline blood flow to40-80%, and the complete occlusion means the reduction of baseline bloodflow to 100%.

The blood flow in the blood vessel may be measured by the method knownin the art, and examples thereof may include ultrasound dilution (Lee KH, Park J Y, Choi S J, Kim J K, Hwang S D, Joh J H, Clinical utility ofaccess blood flow measurement by ultrasound dilution in hemodialysispatients. Korean J Nephrol, 24: 265-273(2005)), doppler ultrasonography(Strauch B, O'Connoll R, Geoly K, Forcasting thrombosis of vascularaccess with doppler flow imaging. Am J Kidney Dis, 19: 554-557, (1992)),glucose pump test (GPT; Magnasco A, Alloatti S, Martinoli C, Solari P,Glucose pump test: a new method for blood flow measurements. NephrolDial Transplant, 17: 2244-2248(2002)), the use of near-infrared(700-1000 nm) (Buunk G, van der Hoeven J G, Meinders A E, A comparisonof near-infrared spectroscopy and jugular bulb oximetry in comatosepatients resuscitated from a cardiac arrest. Anesthesia, 53:13-19(1998)), thermal diffusion blood flow measurement (Ogata N,Fournier J Y, Imhof H G, et al., Thermal diffusion blood flow monitoringduring aneurysm surgery. Acta Neurochir (Wien), 138: 726-731(1996)), andthe like, but are not limited thereto. According to a certain embodimentof the present invention, the blood flow may be measured by using adoppler ultrasonography using ultrasonic waves.

According to a certain embodiment of the present invention, the animalsto which the composition of the present invention is applied are notparticularly limited, and may specifically be mammals; morespecifically, a human being, a mouse, a rat, a rabbit, a monkey, a pig,a horse, a cow, a sheep, an antelope, a dog, and a cat; and still morespecifically, a human and a mouse.

According to a certain embodiment of the present invention, the animalblood vessel includes arteries, veins, and capillaries; morespecifically, main arteries, carotid arteries, subclavian arteries,celiac arteries, mesenteric arteries, renal arteries, iliac arteries,arterioles, capillaries, and venulas; and most preferably main arteriesand carotid arteries.

Diseases that may be treated by the composition of the present inventioninclude a variety of vascular stenosis or occlusive disease, forexample, cerebrovascular disease (CVD), cardiovascular disease,arteriovascular disease, coronary artery disease (CAD), peripheralartery disease (PAD), more specifically, stroke, cerebral infarction,cerebral thrombosis, cerebral embolism, and the like; more specifically,stroke, cerebral infarction, cerebral thrombosis, cerebral embolism,lacunar infarction, acute coronary syndrome, angina, aortic stenosis,myocardial infarction, migraine, bundle branch block, ischemia, acuteischemic arteriovascular event, thrombophlebitis, venousthromboembolism, deep vein thrombosis, pulmonary embolism, peripheralvascular disease, vascular headache, atherosclerosis, vascular spasm,restenosis, restenosis after balloon angioplasty, and vascular occlusionby vasculitis; and most specifically, stroke, cerebral infarction,cerebral thrombosis, cerebral embolism, myocardial infarction.

As used herein, the term “cerebrovascular disease (CVD)” refers toarteriosclerotic vessel disease occurring in the blood vessel, whichsupplies oxygen-rich blood to the face and brain, and thecerebrovascular diseases generally includes comorbid disease occurringtogether with CAD and/or peripheral artery disease (PAD), as well asischemic disease or lack of blood flow. For example, CVD includesischemic cerebrovascular disease, acute ischemic stroke, ischemicstroke, hemorrhagic stroke, varicose veins, mild cognitive impairment(MCI) or transient ischemic attacks (TIA), but is not limited thereto.

As used herein, the term “cardiovascular disease” or “arterioscleroticdisease” is a generic term used to classify numerous conditions thataffect the heart, heart valves, blood, and vasculature, and coversdiseases that affect the heart or blood vessels. Specifically, thisdisease includes metabolic syndromes, syndrome X, atherosclerosis,atherothrombosis, coronary artery disease, stable and unstable angina,stroke, aortic disease, such as aortic stenosis or aortic aneurysm,cerebrovascular disease, peripheral vascular disease, or acute ischemicatherosclerotic events, but is not limited thereto. Especially, thearteriovascular disease means ishemic disease or proischemic disease,rather than non-ishemic disease.

As used herein, the term “coronary artery disease (CAD)” refers toarteriosclerotic vessel disease caused by the hardening and/or narrowingof the artery (coronary artery), which supplies blood to heart muscles,due to atherosclerotic or calcium precipitation. CAD causes a reductionin the blood flow to the heart muscles, such that the heart muscles donot receive a sufficient amount of oxygen, resulting in necrosis. CADincludes acute coronary artery syndrome, myocardial infarction (heartattack), angina (stable and unstable), or atherosclerosis andatherothrombosis, which are caused in blood vessels supplying blood tothe heart, but is not limited thereto.

As used herein, the term “peripheral artery disease (PAD)” refers todisease, such as atherosclerosis or atherothrombosis, occurring in partsother than the heart and brain, and generally includes comorbid diseaseoccurring together with CAD.

As used herein, the term “pharmaceutically effective amount” refers toan amount that is sufficient to attain efficacy or activity (that is,thrombolytic activity) of the above-described saxatilin derivative.

The pharmaceutical composition of the present invention contains apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier contained in the pharmaceutical composition of the presentinvention is conventionally used for the formulation, and examplesthereof may include, but are not limited to, lactose, dextrose, sucrose,sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate,gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, andmineral oil. The pharmaceutical composition of the present invention mayfurther contain a lubricant, a wetting agent, a sweetening agent, aflavoring agent, an emulsifier, a suspending agent, a preservative, andthe like, in addition to the above ingredients. Suitablepharmaceutically acceptable carriers and preparations are described indetail in Remington's Pharmaceutical Sciences (19th ed., 1995).

A suitable dose of the pharmaceutical composition of the presentinvention may vary depending on various factors, such as the method forformulation, manner of administration, the age, body weight, gender, andmorbidity of the patient, diet, time of administration, excretion rate,and response sensitivity. The pharmaceutical composition of the presentinvention may be administered orally or parentally, and examples of theparental administration may include bolus injection, intravenousinjection, subcutaneous injection, intramuscular injection,intraperitoneal injection, dermal administration, etc., and mostspecifically, the pharmaceutical composition may be administered by adirect injection into the blood vessel (e.g., bolus injection orintravenous injection). Direct injection into the blood vessel means anadministration into blood vessels, including arteries, veins, andcapillaries, for example, main arteries, carotid arteries, subclavianarteries, celiac arteries, mesenteric arteries, renal arteries, iliacarteries, arterioles, capillaries, and venulas, and the administrationmanner may be selected appropriately according to the vascular portionin which the thrombus is generated.

In addition, the route of administration of the pharmaceuticalcomposition of the present invention is preferably determined accordingto the disease type, to which the composition is applied, and thepractitioner. For example, the concentration of the saxatilinderivative, which is an active ingredient contained in the compositionof the present invention, may be determined considering the therapeuticpurpose, the condition of the patient, the required period, or the like,and is not limited to a specific range of concentration. According to acertain embodiment of the present invention, the daily dose of thepharmaceutical composition of the present invention is 0.001-1,000mg/kg.

The pharmaceutical composition of the present invention is formulatedinto a unit dosage form or in a multidose container, using apharmaceutically acceptable carrier and/or excipient according to themethod that can be easily conducted by a person having ordinary skillsin the art to which the present invention pertains. Here, the dosageform may be a solution in an oily or aqueous medium, a suspension, anemulsion, an extract, a powder, a granule, a tablet, or a capsule, andmay further include a dispersant or a stabilizer.

The pharmaceutical composition of the present invention is characterizedin that the vascular stenosis or occlusive disease is treated not by theprevention or treatment through a simple principle of thrombusgeneration prevention, but by a principle in which the already generatedthrombus is lysed to penetrate the blood vessel effectively. Forexample, in the case of stroke, the pharmaceutical composition (e.g.,aspirin) that prevents thrombus generation has been already well known.However, once cerebral infarction occurs by thrombus generation andbrain blood vessel occlusion, there are no pharmaceutical compositionsthat may effectively treat the cerebral infarction. The treatment byintravenous administration of recombinant tissue plasminogen activator(r-tPA) is currently the only approved therapy for cerebral infarctionwithin 3 hours of symptom onset. However, the treatment using theplasminogen activator is constrained by time, and in a situation inwhich more than half of the patients fail to achieve successfulrecanalization after thrombolytic treatment, the present pharmaceuticalcomposition, which can effectively lyse the already generated thrombi,is a very realistic and groundbreaking approach in the treatment ofvascular stenosis or occlusive disease that has already occurred. Notonly that, the pharmaceutical composition for vascular stenosis orocclusive disease of the present invention is of great value in that iteffectively also treats microvascular occlusion and causes norestenosis.

In accordance with another aspect of the present invention, there isprovided a method for preventing or treating vascular stenosis orocclusive disease, the method including administering a pharmaceuticallyeffective amount of the saxatilin derivative to a subject.

In an embodiment of the present invention, the vascular stenosis orocclusive disease is selected from the group consisting of stroke,cerebral infarction, cerebral thrombosis, cerebral embolism, lacunarinfarction, acute coronary syndrome, angina, aortic stenosis, myocardialinfarction, migraine, bundle branch block, ischemia, acute ischemicarteriovascular event, thrombophlebitis, venous thromboembolism, deepvein thrombosis, pulmonary embolism, peripheral vascular disease,vascular headache, atherosclerosis, vascular spasm, restenosis,restenosis after balloon angioplasty, and vascular occlusion byvasculitis.

Since the method for the prevention or treatment of the presentinvention is achieved by administering to a subject the “saxatilinderivative” according to another aspect of the present invention,descriptions of overlapping contents between the two are omitted toavoid excessive complication of the specification due to repetitivedescriptions thereof.

In accordance with another aspect of the present invention, there isprovided a use of a saxatilin derivative for preventing or treatingvascular stenosis or occlusive disease.

Since the use of the present invention is directed to the use of the“saxatilin derivative” according to another aspect of the presentinvention, descriptions of overlapping contents between the two areomitted to avoid excessive complication of the specification due torepetitive descriptions thereof.

Advantageous Effects

Features and advantages of the present invention are summarized asfollows:

(a) The present invention relates to a saxatilin derivative having anincreased half-life and a use thereof.

(b) The saxatilin derivative of the present invention has similarthrombolytic activity to the parent protein saxatilin as well as asignificantly increased protein half-life, and enables very efficientlysis of the thrombi, which were already generated in the blood vesselin FeCl3-induced carotid artery animal models, using the saxatilinderivative.

(c) Therefore, the composition containing the saxatilin derivative as anactive ingredient, of the present invention, causes no restenosis aftercanalization and effectively opens even microvessels, and thus thecomposition is very effective in the treatment of vascular stenosis orocclusive diseases (e.g., cerebrovascular diseases, cardiovasculardiseases, arteriosclerotic vascular diseases, coronary artery diseases,and peripheral vascular diseases).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows structures of saxatilin derivatives that arecloned to multicloning sites of pYK603 vector and pYK602 vector,respectively. The distance between components or the distance betweenrestriction enzymes is not drawn to the scale. leader (leader sequence),immunoglobulin G2a gamma chain.

FIG. 2 shows western blotting results confirming the expression of thesaxatilin derivatives of the present invention. For verifying thestability of purified proteins, the presence or absence of precipitateswas checked by centrifugation at 5,000 rpm for 10 minutes (lane 2 andlane 4). As a result, the saxatilin derivatives of the present inventionwere very stable (no precipitations).

FIGS. 3A and 3B show purification results of Fc-saxatilin andsaxatilin-Fc, respectively. Gel-electrophoresis images (left panels) andelectropherograms (right panels) are presented.

FIG. 4 shows measurement results of platelets aggregation-inhibitoryactivities of saxatilin, as a parent protein, and the saxatilinderivatives of the present invention (Fc-saxatilin and saxatilin-Fc).For platelet aggregation assay, saxatilin, as a parent protein, andFc-saxatilin were used at 100, 150, 200, 250, and 300 nM, andsaxatilin-Fc was used at 200, 300, 350, 400, 500, and 600 nM.

FIGS. 5A to 5C shows measurement results of half-lives of saxatilin, asa parent protein, and the saxatilin derivatives (Fc-saxatilin andsaxatilin-Fc) of the present invention.

FIG. 6 shows thrombolytic effect comparison results between thesaxatilin derivative and 650 nmol/kg saxatilin. In addition, thepresence and degree of recanalization were determined by measuring theblood flow.

FIG. 7 shows a dose-response curve of the mean values of each group thatwere obtained in a dose-response study. It shows comparison results withthe thrombolytic effects of 650 nmol/kg saxatilin (mean; red line;standard deviation; red area), which were obtained in the previousstudy.

FIG. 8 shows the mean blood flow percent to the baseline blood flow, forthe last 10 minutes of 2 hours of the measurement of thrombolyticeffect.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail withreference to examples. These examples are only for illustrating thepresent invention more specifically, and it will be apparent to thoseskilled in the art that the scope of the present invention is notlimited by these examples.

Examples Methods and Results

Construction, Expression, and Purification of Recombinant SaxatilinProtein Expression Vectors

Construction of Fc-Saxatilin and Saxatilin-Fc Protein Expression Vectors

For the expression of the recombinant saxatilin protein, the presentinventors conducted PCR using the primer pair (saxatilin-F andsaxatilin-R) shown in the table below. The obtained PCR products werecleaved with restriction enzyme sfil (Thermo Scientific), and clonedinto pYK603 vector and pYK602 vector (A&R Therapeutics) using T4 DNAligase (Thermo Scientific), thereby constructing recombinant vectors forexpressing Fc-saxatilin protein and saxatilin-Fc protein.

TABLE 1 Primer sequences for expression vector of Fc-saxatilin (restriction enzyme cleavage  sequences, underlined) Primername sequence(5′->3′) saxa- agta ggccgtgggggcc gaggccggagaagaatgtgactgttilin-F saxa- gcat ggccgacgcggcc aaggcatggaagggatttctgggaca tilin-R

Confirmation of Fc-Saxatilin and Saxatilin-Fc Protein Expression

292E cells were transfected with the foregoing recombinants vectorscontaining Fc-saxatilin and saxatilin-Fc using polyethylenimine (PEI,Polysciences), respectively. After the transfection, each supernatantwas collected on day 2 or 5 of cell culture, subjected to SDS-PAGE,transferred to NC membrane (Millipore), and then allowed to react with asecondary antibody, anti-HuFc-HRP (1:2000 dilution, Thermo Scientific)at room temperature for 1 h. The NC membrane was washed three times withPBS/T, and then western blotting was conducted through ECL solutiontreatment and film development (FIG. 2).

Purification of Fc-Saxatilin and Saxatilin-Fc Proteins

On day 3, 5, or 7 of culture of the transfected 293E cells, each culturesupernatant was filtered through 0.22-μm top-filter (Millipore). Thefiltered supernatant was loaded through 5 ml column packed with 500 μlof protein A beads (GE healthcare) to allow to bind with the beads. Thebinding reaction was conducted at 4° C. overnight at a flow rate of 0.9ml/min using a peri-start pump (Bio-rad), followed by washing with 100ml or more of PBS, and then six fractions were eluted using 0.1 Mglycine-HCl. The eluates were neutralized with 1 M Tris (pH 9.0)solution. Thereafter, protein quantification was conducted, and theproteins contained in 2-3 fractions were concentrated using Amicon Ultracentrifugal filter (Millipore). Buffer was exchanged ten times or morewith PBS.

The purity of the obtained protein concentrate was analyzed usingAgilent 2100 bioanalyzer (FIGS. 3A and 3B). The Agilent 2100 bioanalyzeris a system that can automatically analyze both the size andconcentration of proteins based on a lab-on-a-chip. The whole-processesof sample processing, isolating, and detecting, were simultaneouslyperformed in microchips, thereby obtaining highly reliable data, ofwhich the analysis results were then confirmed by two types(gel-electrophoretic imaging, and an electropherogram such aschromatography).

Platelet Aggregation-Inhibitory Activity of Fc-Saxatilin andSaxatilin-Fc Proteins

Anesthetic solution was prepared by mixing zoletil 50 (VirBac) andrompun (Bayer) at a ratio of 3:1 and then 10-fold diluting the mixturewith physiological saline. Female Institute of Cancer Research (ICR)mice (Orientbio Inc, Korea) were weighed, and 10 μl/g per g of theanesthetic solution was intraperitoneally injected. The anesthetizedmouse was fixed and the abdomen part is cut open, and then the blood wascollected and put in a sodium citrate tube (#363048; BD Science),followed by well stirring. The collected blood was transferred to a 15ml tube, and centrifuged at 1,000 rpm for 15 minutes to obtainplatelet-rich plasma (PRP). In addition, the lower hemocyte layer wasagain centrifuged at 3,000 rpm for 10-15 minutes to obtain supernatant(plasma). The supernatant (600 μl) was centrifuged at 10,000 rpm for 2minutes to obtain hemocytes without platelet-poor plasma (PPP), and theremaining plasma was mixed with the PRP, which was separated in theprevious step, before use. Platelet aggregation assay was conductedusing an aggregometer (Model 700; Chrono-log). A first cuvette (#312;Chrono-log) added with PPP (500 μl), and a second cuvette added with PRP(480 μl) and saxatilin (10 μl) or recombinant saxatilin (Fc-saxatilin orsaxatilin-Fc; 10 μl) were put in the aggregometer to set the baseline.Then, 10 μl of ADP (final concentration, 20 μM; #384; Chrono-log) wasput in the second cuvette, followed by mixing through pipetting, and theaggregation degree was measured. Here, assuming that the aggregationdegree was 100% for only ADP, on the basis of which the aggregationdegree of each sample was calculated as a relative value. That is, thevalue obtained by subtracting the aggregation rate (%) of each samplefrom 100% was calculated as an inhibition (%). The IC₅₀ concentration ofnaturally occurring saxatilin with respect to platelet aggregation was150 nM, and the IC₅₀ concentrations of Fc-saxatilin and saxatilin-Fcwere 196 nM and 362 nM, respectively (FIG. 4), and these results showthat the recombinant saxatilin of the present invention exhibitedexcellent platelet aggregation activity, which can be compared with thatof saxatilin.

Half-Lives of Fc-Saxatilin and Saxatilin-Fc Proteins

N-hydroxysuccinimide-rhodamine (NHS-Rhodamine; #46406, 10 mg/ml; ThermoScientific Pierce, USA) was added to reaction tubes containingsaxatilin, Fc-saxatilin, and saxatilin-Fc (in PBS, pH 7.4), followed bya reaction at room temperature for 1 hour. Non-reacted NHS-Rhodamine wasremoved using PD10 salting column (#17-0851-01; GE Healthcare, USA). Theconcentrations of rhodamine-conjugated proteins were measured using BCAprotein assay kit (#23225; Thermo Scientific Pierce, USA), and then theproteins were stored at 4° C. in dark conditions until used forexperiments.

Rhodamine-labeled saxatilin was diluted with PBS buffer to 0.5 mg/ml,and then injected to 8-week aged ICR mice (three mice; Orientbio Inc,Korea) through the tail vein using an insulin injector (#32882; BDScience, USA). Here, the doses were 5 mg/10 ml/kg (mouse weight). Theblood was collected from the jugular vein of the mice 0 min, 5 min, 10min, 20 min, 40 min, and 80 min after the administration of the protein,and was maintained at room temperature in dark conditions for 30 min,leading to blood coagulation. The blood was centrifuged at 12,000 rpmfor 10 min at 4° C. to separate supernatant (serum). 80 μl of theobtained serum was put in dark 96-well plate (#43711; Thermo ScientificPierce, USA), and subjected to fluorescence measurement using amicroplate fluorometer (excitation wavelength of 552 nm and emissionwavelength of 575 nm; Thermo Scientific Pierce, USA), therebycalculating the half-life.

The rhodamine-conjugated Fc-saxatilin or saxatilin-Fc was diluted withPBS buffer to 0.5 mg/ml, and then injected to 9-week aged ICR mice (fivemice) through the tail vein using an insulin injector. Here, the doseswere 5 mg/10 ml/kg (mouse weight). The blood was collected from thejugular vein of the mice 0 min, 5 min, 10 min, 20 min, 30 min, 60 min, 2hr, 4 h, and 8 h after the administration of the protein, and wasmaintained at room temperature in dark conditions for 30 min, leading toblood coagulation. The blood was centrifuged at 12,000 rpm for 10 min at4° C. to separate supernatant (serum). 80 μl of the obtained serum wasput in dark 96-well plate (#43711; Thermo Scientific Pierce, USA), andsubjected to fluorescence measurement using a microplate fluorometer(excitation wavelength of 552 nm and emission wavelength of 575 nm),thereby calculating the half-life.

As a result, the half-lives of the rhodamine-conjugated saxatilin,Fc-saxatilin, and saxatilin-Fc were 2.0 min, 8.6 min, and 12.5 min,respectively (FIGS. 5A to 5C). Therefore, it was confirmed that theFc-saxatilin and saxatilin-Fc of the present invention had significantlyincreased half-lives compared with saxatilin.

Binding Between Integrin and Saxatilin

Integrins (α_(II) _(b) β₃, α_(V)β₃, α_(M)β₂, and α_(L)β₂) were added to96-well plate at 100 ng/well, respectively, followed by coating at 4° C.overnight. After the coating, the wells were washed three times withPBS-T (0.5% Tween 20). The wells were blocked with 1% BSA (in PBS-T)solution at room temperature for 2 h. After the blocking, the wells werewashed three times with PBS-T. Saxatilin-Fc or Fc-saxatilin was dilutedserially 4-fold from 100 nM to prepare eight sample solutions.

100 μl of the sample solutions were triply loaded to each well, followedby a reaction at room temperature for 2 h. After the reaction, the wellswere washed three times with PBS-T. 100 μl of anti-human IgG(Fc)-conjugated HRP antibodies, diluted to 1:20,000, was added to eachwell, followed by a reaction at room temperature for 1 h. After thereaction, the wells were washed four times with PBS-T. TMB solution wasadded, followed by a reaction at room temperature for 30 min, and thenthe reaction was stopped by adding a stop solution (0.5 M H₂SO₄). Theabsorbance was measured at 450 nm to verify the binding betweensaxatilin and integrins, involved in an interaction between neutrophilsand endothelial cells.

Integrins α_(M)β₂ and α_(L)β₂, which exist in the neutrophils, interactwith intercellular adhesion molecule 1 (ICAM-1) in endothelial cells toallow the neutrophils to gather in the endothelial cells. As a result ofbinding analysis using ELISA, Fc-saxatilin strongly bound with integrinsα_(M)β₂ and α_(L)β₂ (Kd, 6.9×10⁻⁹ M and 9.2×10⁻⁹ M). In addition,although more weakly than Fc-saxatilin, the saxatilin-Fc bound withintegrins α_(M)β₂ and α_(L)β₂ (Kd, 3.0×10⁻⁷ M and 4.4×10⁻⁷ M) (FIG. 9).

Thrombolytic Effect of Recombinant Saxatilin Derivatives UsingThrombolysis Model by Fecl₃

8-week aged female ICR mice with 32-34 g were used. Animal care and usewere performed according to protocols reviewed and approved by theInstitutional Animal Care and Use Committee (IACUC) at the YonseiUniversity College of Medicine on the basis of the standards suitable tothe guide of the Association for Assessment and Accreditation ofLaboratory Animal Care, International (AAALAC). For surgical operation,animals were anesthetized through breathing with 5% isoflurane in amixture (Hankook Special Gases, Korea) composed of 70% N₂O and 30% O₂.The anesthetization was maintained with 2% isoflurane. During thesurgical operation, the animal body temperature was continuouslymonitored using a probe, and maintained at 37.0±0.2° C. using ahomeothermic blanket control unit and heating pad (Harvard Apparatus,Holliston, Mass.). In order to test in vivo thrombolytic activity ofsaxatilin and saxatilin-Fc, FeCl₃ (Sigma-Aldrich, USA)-induced carotidartery thrombus models were used. A midline cervical incision was madeto carefully anatomize the common carotid artery (CCA) under anoperating microscope (SEILER, USA). An ultrasonic doppler flow probe(Transonic MA0.7PSB) was positioned at the center portion of the commoncarotid artery (CCA). The carotid blood flow was measured using anultrasonic TS420 blood flowmeter (Transonic Instruments, Ithaca, N.Y.)and an iWorx IX-304T data acquisition system (iWorx Systems, Inc.,Dover, N.H.). The baseline blood flow of CCA was measured for 5 min fora control. After the determination of the baseline blood flow of thecontrol, the probe was removed. Oxidative vascular injury by chemicalstress was induced by placing a filter paper (700×500 μm) saturated with50% FeCl₃ on the adventitial surface of the midpoint of the exposed CCAfor 5 min. After removing the filter paper, the CCA was washed withphysiological saline, and the blood flow thereof was measured. Thrombusgeneration and arterial occlusion were determined by a decrease in theblood flow, and the complete occlusion was defined as an absence of theblood flow for 10 minutes.

Ten minutes after occlusion of the CCA, Fc-saxatilin was bolus-injectedat 0, 1.5, 7.5, 15, and 150 nmol/kg through the left femoral vein byusing an infusion pump (KD Scientific Inc., USA) connected to PE-10tubing (Becton Dickinson and Company, USA). The carotid blood flow wascontinuously monitored for 2 hours from the initial time of injection.The thrombolytic effect was evaluated using five mice per group, andcompared with the effect of 650 nmol/kg saxatilin, which was evaluatedin a previous study.

In addition, the presence and degree of recanalization were determinedby measuring the blood flow (FIG. 6B). Data about the baseline bloodflow and the blood flow monitored continuously for 2 h after CCAocclusion were obtained using iWorx Labscribe2 data acquisition software(version 2.045000). The carotid blood flow was analyzed by calculatingthe area under the obtained flow-time curve. All the measured valueswere standardized by the minimum blood flow of each animal to avoiddifferences caused by the variation in physiological condition betweenanimals. The thrombolytic effect was calculated as below, and expressedas a percent of mean baseline blood flow: (mean blood flow for 2 h aftersaxatilin derivative administration/mean baseline blood flow)×100(%). Indose-response study, the mean values of each group were calculated andexpressed by a dose-response curve (mean±SD, FIG. 7). In addition, theabove results were compared with the thrombolytic effects (mean; redline; standard deviation; red region) of 650 nmol/kg saxatilin, whichwas obtained in the previous study). Meanwhile, the mean blood flow ofevery 1 min was calculated in each animal, and respective representativepatterns of dose and administration method were checked in adose-dependent manner (FIG. 6A). The mean values of all animals in therespective group were calculated, and the temporal changes were shownthrough continuous bar graphs (mean±SD).

When compared with the baseline blood flow, the mean percents of bloodflow in administration groups were as follows (FIG. 7): (a)physiological saline administered group, 3.03±0.75%; (b) Fc-saxatilin1.5 nmol/kg administered group, 17.81±12.22%; (c) Fc-saxatilin 7.5nmol/kg administered group, 89.40±41.18%; (d) Fc-saxatilin 15 nmol/kgadministered group, 59.94±16.97%; (e) Fc-saxatilin 150 nmol/kgadministered group, 74.68±20.53%; and (f) saxatilin 650 nmol/kgadministered group, 78.24±48.61% (mean±SD).

The mean blood flow percent to the baseline blood flow, for the last 10minutes of 2 hours of the measurement of thrombolytic effect, wasanalyzed (FIG. 8). (a) physiological saline administered group,3.29±1.25%; (b) Fc-saxatilin 1.5 nmol/kg administered group,23.06±11.75%; (c) Fc-saxatilin 7.5 nmol/kg administered group,119.52±46.32%; (d) Fc-saxatilin 15 nmol/kg administered group,69.05±26.07%; (e) Fc-saxatilin 150 nmol/kg administered group,72.49±22.49%; and (f) Fc-saxatilin 650 nmol/kg administered group,81.54±57.50% (mean±SD).

In summary, Fc-saxatilin showed a dose-dependent thrombolytic effect.The mean thrombolytic effect and the thrombolytic effect for last 10minutes by Fc-saxatilin 7.5 nmol/kg, 15 nmol/kg, and 150 nmol/kgadministration showed statistically similar effects compared withexisting saxatilin 650 nmol/kg.

In the case where 650 nmol/kg saxatilin was administered, the saxatilinadministered group showed clear decreases in the thrombolytic effect andthe thrombus reformation inhibitory effect with the passage of time. Theabove-described abrupt reocclusion was observed in three of a total offive mice, into which saxatilin was bolus-injected, for above 90 minuteson average (the arrow in FIG. 6A). Whereas, it was confirmed that, incases where Fc-saxatilin was administered in 7.5 nmol/kg or more, thethrombolytic effect and the thrombus formation inhibitory effect wereconstantly maintained for 2 hours without reocclusion afterrecanalization.

Although the present invention has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only for a certain embodiment anddoes not limit the scope of the present invention. Thus, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

REFERENCES

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What is claimed is:
 1. A saxatilin derivative comprising saxatilin,which is composed of the amino acid sequence of SEQ ID NO: 2, conjugatedto an immunoglobulin Fc region.
 2. The saxatilin derivative of claim 1,wherein the immunoglobulin Fc region is conjugated to the N-terminal orC-terminal of the saxatilin.
 3. The saxatilin derivative of claim 1,wherein the saxatilin derivative comprises a leader sequence furtherconjugated to the N-terminal.
 4. The saxatilin derivative of claim 1,wherein the saxatilin derivative has a half-life, which is increased by4- to 6.5-fold when compared with naturally occurring saxatilin.
 5. Thesaxatilin derivative of claim 1, wherein the saxatilin derivative has asimilar thrombolytic ability to naturally occurring saxatilin.
 6. Thesaxatilin derivative of claim 5, wherein the saxatilin derivative has anIC₅₀ value of 100-500 nM, with respect to platelet aggregation. 7.(canceled)
 8. The saxatilin derivative of claim 1, wherein the saxatilinderivative binds to integrins existing in a thrombus to break up thethrombus.
 9. The saxatilin derivative of claim 8, wherein the saxatilinderivative binds to glycoprotein (GP) Ilb-Ilia on the surface ofplatelets constituting the thrombus to break up the thrombus.
 10. Thesaxatilin derivative of claim 1, wherein the saxatilin derivative has abinding affinity (dissociation constant (Kd)) of 1×10⁻⁸ to 1×10⁻¹⁰ M tointegrin α_(M)β₂ existing in neutrophils.
 11. The saxatilin derivativeof claim 1, wherein the saxatilin derivative has a binding affinity(dissociation constant (Kd)) of 1×10⁻⁸ to 1×10⁻¹⁰ M to integrin α_(L)β₂existing in neutrophils. 12-15. (canceled)
 16. A pharmaceuticalcomposition for preventing or treating vascular stenosis or occlusivedisease, the pharmaceutical composition containing: (a) apharmaceutically effective amount of the saxatilin derivative of claim1; and (b) a pharmaceutically acceptable carrier.
 17. The pharmaceuticalcomposition of claim 16, wherein a blood vessel in the vascular stenosisinclude main arteries, carotid arteries, subclavian arteries, celiacarteries, mesenteric arteries, renal arteries, iliac arteries,arterioles, capillaries, and venulas.
 18. The pharmaceutical compositionof claim 16, wherein the vascular stenosis or occlusive disease isselected from the group consisting of stroke, cerebral infarction,cerebral thrombosis, cerebral embolism, lacunar infarction, acutecoronary syndrome, angina, aortic stenosis, myocardial infarction,migraine, bundle branch block, ischemia, acute ischemic arteriovascularevent, thrombophlebitis, venous thromboembolism, deep vein thrombosis,pulmonary embolism, peripheral vascular disease, vascular headache,atherosclerosis, vascular spasm, restenosis, restenosis after balloonangioplasty, and vascular occlusion by vasculitis.
 19. Thepharmaceutical composition of claim 16, wherein the pharmaceuticalcomposition is administered through a direct injection into the bloodvessel.
 20. The pharmaceutical composition of claim 16, wherein thepharmaceutical composition is administered to a patient with vascularocclusion due to thrombi.
 21. The pharmaceutical composition of claim20, wherein the occlusion is partial occlusion or complete occlusion.22. The pharmaceutical composition of claim 16, wherein thepharmaceutical composition contains no plasminogen activator.
 23. Amethod for preventing or treating vascular stenosis or occlusivedisease, the method comprising administering a pharmaceuticallyeffective amount of the saxatilin derivative of claim 1 to a subject.24. The method of claim 23, wherein the vascular stenosis or occlusivedisease is selected from the group consisting of stroke, cerebralinfarction, cerebral thrombosis, cerebral embolism, lacunar infarction,acute coronary syndrome, angina, aortic stenosis, myocardial infarction,migraine, bundle branch block, ischemia, acute ischemic arteriovascularevent, thrombophlebitis, venous thromboembolism, deep vein thrombosis,pulmonary embolism, peripheral vascular disease, vascular headache,atherosclerosis, vascular spasm, restenosis, restenosis after balloonangioplasty, and vascular occlusion by vasculitis.